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The effects of hydration on the DNA conformation in the colon biopsy tissues at different disease stages, hyperplasia, dysplasia, and cancer, and their subsequent classifications were investigated in this study. FTIR spectroscopic imaging was used to study the tissues while controlling the humidity from 16% RH to 88% RH using saturated salt solutions. A nonuniform uptake of water into the tissue at its maximum hydrated state was observed in the chemical images showing the distribution of the absorbance of the νas OH spectral band. The regions of high absorbance of this band in the tissues overlap with the regions of high absorbance of predominantly the phosphate (1143-1100 cm-1) and lipid (2879-2844 cm-1) bands. Analysis of the second derivative spectra of the hydrated and dehydrated colon tissues further revealed significant peak shifts and changes in absorbance of the spectral bands that correspond to the vibrations of the phosphate group of DNA. These findings showed some disparities when compared to the effect of hydration on the infrared spectra of live cells and pure isolated DNA, possibly due to the presence of DNA mostly in its A-form in the formalin fixed tissues. Coupled with principal component analysis, the spectral biomarkers that differentiate the healthy colon tissues from the diseased tissues were identified to be in the phosphodiester spectral region (1300-1000 cm-1). This differentiation varied under different humidity conditions, with the highest sensitivity of ∼98% found at the dehydrated state of the tissues with random forest supervised classification.

In this study, the novel application of ATR-FTIR spectroscopy and macro ATR-FTIR spectroscopic imaging overcame an analytical challenge in conservation science: the time-resolved, chemical, and spatial investigation of the reaction of inorganic treatments for stone conservation (ammonium oxalate, AmOx; ammonium phosphate, DAP) occurring in water-based solutions. The aim was to (1) assess the composition and localization of reaction products and their phase variation during the reaction in real time and directly in an aqueous environment and (2) investigate the reaction of AmOx and DAP with calcite and the transformations induced to the substrate with a time-resolved approach. The new analytical results showed that for both treatments, the formation of new crystalline phases initiated at the early stages of the reaction. Their composition changed during the treatment and led to more stable phases. The reactivity of the stone substrate to the treatments varied as a function of the stone material features, such as the specific surface area. A clear influence of post-treatment rinsing on the final composition of reaction phases was observed. Above all, our research demonstrates the actual feasibility, practicality, and high potential of an advanced ATR-FTIR spectroscopic approach to investigate the behavior of conservation treatments and provided new analytical tools to address the choices of conservation in pilot worksites. Lastly, this study opens novel analytical perspectives based on the new possible applications of ATR-FTIR spectroscopic imaging in the field of conservation science, materials science, and analytical chemistry.

Monoclonal antibodies (mAbs) have been used as therapeutics for the last few decades. It is necessary to investigate the stability of these mAbs under stress conditions and to elucidate aggregation mechanisms as a means of developing approaches which minimize the problem. Attenuated total reflection (ATR)-FTIR spectroscopic imaging allows probing of a sample at a depth of penetration of around 0.5-5 μm, which makes it suitable for the study of aggregated proteins when accumulated as a layer close to the surface of the ZnSe internal reflection element (IRE). Here, macro ATR-FTIR spectroscopic imaging, along with a variable angle of incidence accessory, have been used to differentiate between the secondary structure of proteins in bulk solution and those that have precipitated onto or near the ZnSe IRE surface. IgG spectra obtained from protein samples in individual wells have been averaged, extracted, and preprocessed, and the Amide I bands of the protein samples were compared and further analyzed to reveal protein distribution at the ZnSe IRE surface. These findings show depth profiling of IgG aggregates at the ZnSe IRE surface (0.5-5 μm) and do not follow a trend of decreasing protein presence with an increasing angle of incidence or increasing depth of penetration, suggesting an irregular distribution of aggregates in the z-direction.

The first analytical intercomparison of fingerprint residue using equivalent samples of latent fingerprint residue and characterized by a suite of relevant techniques is presented. This work has never been undertaken, presumably due to the perishable nature of fingerprint residue, the lack of fingerprint standards, and the intradonor variability, which impacts sample reproducibility. For the first time, time-of-flight secondary ion mass spectrometry, high-energy secondary ion mass spectrometry, and X-ray photoelectron spectroscopy are used to target endogenous compounds in fingerprints and a method is presented for establishing their relative abundance in fingerprint residue. Comparison of the newer techniques with the more established gas chromatography/mass spectrometry and attenuated total reflection Fourier transform infrared spectroscopic imaging shows good agreement between the methods, with each method detecting repeatable differences between the donors, with the exception of matrix-assisted laser desorption ionization, for which quantitative analysis has not yet been established. We further comment on the sensitivity, selectivity, and practicability of each of the methods for use in future police casework or academic research.

Rapid, in situ, and label-free chemical analysis in microfluidic devices is highly desirable. FT-IR spectroscopic imaging has previously been shown to be a powerful tool to visualize the distribution of different chemicals in flows in a microfluidic device at near video rate imaging speed without tracers or dyes. This paper demonstrates the possibility of using this imaging technology to capture the chemical information of all reactants and products at different points in time and space in a two-phase system. Differences in the rates of chemical reactions in laminar flow and segmented flow systems are also compared. Neutralization of benzoic acid in decanol with disodium phosphate in water has been used as the model reaction. Quantitative information, such as concentration profiles of reactant and products, can be extracted from the imaging data. The same feed flow rate was used in both the laminar flow and segmented flow systems. The laminar flow pattern was achieved using a plain wide T-junction, whereas the segmented flow was achieved by introducing a narrowed section and a nozzle at the T-junction. The results show that the reaction rate is limited by diffusion and is much slower with the laminar flow pattern, whereas the reaction is completed more quickly in the segmented flow due to better mixing.

This work introduces a new and nondestructive methodology for the collection and chemical identification of latent fingermarks. The main challenges of this work were (a) to find an appropriate medium to lift fingermarks from various surfaces and (b) to develop an analytical approach for the identification of small quantities of sample while avoiding spectroscopic interference from the lifting media. Two different lifting media were evaluated and analyzed by ATR-FT-IR spectroscopic imaging, which affords inherent chemical specificity with rapid acquisition of data. This is the first time that chemical images of latent fingermarks collected with gel lifters from different surfaces have been obtained. Spatially resolved chemical images from different depths within the same sample were obtained using ATR-FT-IR imaging with a variable angle ATR accessory to minimize interference from the substrate. The possibility of obtaining, through the developed methodology, three-dimensional depth profiles of surface contaminants collected with the lifting gel shows great potential for the investigation of samples for forensic interest.

This paper demonstrates an approach to obtain chemical images of pharmaceutical tablets using attenuated total reflection infrared (ATR-IR) spectroscopy. FT-IR images with different fields of view and spatial resolution have been obtained using a combination of different ATR accessories. FT-IR imaging with the diamond ATR accessory and micro-ATR imaging technique have been compared. With the diamond ATR imaging accessory, compaction to a tablet can be performed and the chemical image measured in situ. It has been found that the diamond ATR imaging accessory gives information on the overall distribution of different components in a tablet while the micro-ATR imaging technique provides a closer look at the tablet with 4-microm spatial resolution. Low-concentration components down to 0.5% have been detected by the micro-ATR method. Both experimental and commercial systems are studied in this paper.

The use of biotherapeutics, such as monoclonal antibodies, has markedly increased in recent years. It is thus essential that biotherapeutic production pipelines are as efficient as possible. For the production process, one of the major concerns is the propensity of a biotherapeutic antibody to aggregate. In addition to reducing bioactive material recovery, protein aggregation can have major effects on drug potency and cause highly undesirable immunological effects. It is thus essential to identify processing conditions which maximize recovery while avoiding aggregation. Heat resistance is a proxy for long-term aggregation propensity. Thermal stability assays are routinely performed using various spectroscopic and scattering detection methods. Here, we evaluated the potential of macro attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopic imaging as a novel method for the high-throughput thermal stability assay of a monoclonal antibody. This chemically specific visualization method has the distinct advantage of being able to discriminate between monomeric and aggregated protein. Attenuated total reflection is particularly suitable for selectively probing the bottom of vessels, where precipitated aggregates accumulate. With focal plane array detection, we tested 12 different buffer conditions simultaneously to assess the effect of pH and ionic strength on protein thermal stability. Applying the Finke model to our imaging kinetics allowed us to determine the rate constants of nucleation and autocatalytic growth. This analysis demonstrated the greater stability of our immunoglobulin at higher pH and moderate ionic strength, revealing the key role of electrostatic interactions. The high-throughput approach presented here has significant potential for analyzing the stability of biotherapeutics as well as any other biological molecules prone to aggregation.

Protein crystallization is of strategic and commercial relevance in the post-genomic era because of its pivotal role in structural proteomics projects. Although protein structures are crucial for understanding the function of proteins and to the success of rational drug design and other biotechnology applications, obtaining high quality crystals is a major bottleneck to progress. The major means of obtaining crystals is by massive-scale screening of a target protein solution with numerous crystallizing agents. However, when crystals appear in these screens, one cannot easily know if they are crystals of protein, salt, or any other molecule that happens to be present in the trials. We present here a method based on Attenuated Total Reflection (ATR)-FT-IR imaging that reliably identifies protein crystals through a combination of chemical specificity and the visualizing capability of this approach, thus solving a major hurdle in protein crystallization. ATR-FT-IR imaging was successfully applied to study the crystallization of thaumatin and lysozyme in a high-throughput manner, simultaneously from six different solutions. This approach is fast as it studies protein crystallization in situ and provides an opportunity to examine many different samples under a range of conditions.